The present invention relates generally to apparatus for synthesizing and culturing chemical and biological compounds, to chemical and biological filtration apparatus, and to multi-well apparatus, and more particularly to such apparatus used for performing multiple simultaneous syntheses and filtrations on a micro scale.
As the scale on which chemical compounds and biological materials are capable of being analyzed, tested, purified and otherwise manipulated for research and clinical purposes continues to decrease, there is a call for increasingly more efficient methods of synthesizing and culturing such compounds and materials. Numerous benefits, in particular, are offered by the extremely small or xe2x80x9cmicroxe2x80x9d scale preparation of chemical and biochemical molecules. Among such benefits are that costs with respect to the reagents, solvents and other materials employed for reaction, workup and purificationxe2x80x94many of which can be quite expensivexe2x80x94are greatly reduced. Further, the amounts of by-products and other waste materials generated are also greatly reduced, thereby lowering disposal costs and also reducing the potential for environmental damage by hazardous materials. Moreover, the speed with which large numbers of chemical compounds can be synthesized for biological screening purposes can be greatly increased, since a large number of such microscale reactions can potentially be run and processed simultaneously in a single piece of apparatus using batch equipment.
Given that only a very minute percentage of chemical entities that are investigated will exhibit the desired characteristics for which they have been targeted and synthesized, and given that relatively small changes in chemical structure can produce profoundly different biological properties, the ability to rapidly synthesize large numbers of new compounds and analogs for evaluation is of especially great commercial importance, indeed, it is often a matter of economic survival to pharmaceutical and biotechnological companies involved in the development of new therapeutic agents.
It has been the case for a number of years in the peptide field that use has been made of mechanized, computer-aided equipment capable of simultaneously synthesizing a number of different peptides by the sequential coupling of amino acids to functionalized solid supports. Such mechanized equipment may be employed because the conditions necessary for most such coupling reactions are uniformly simple and straightforward.
In the case of the synthesis of organic compounds generally, however, there is a great variance in the conditions and techniques which must be employed, which precludes or makes impractical the use of fully automated instruments. For example, magnetic stirring, shaking, or some other form of agitation may be needed, and heating or cooling to great temperature extremes may be required. With respect to reactions requiring heating, provision for the reflux of volatile solvent may also be necessary. Further, many chemical reactions are air sensitive and so may require an inert atmosphere for their performance. Reagents and substrates may be air sensitive (e.g., hygroscopic or pyrophoric) or corrosive and require special handling techniques, such as transfer via a syringe or cannula, or even handling in a glove box. Many organic synthetic procedures require the addition of materials in both solid and liquid form or employ an addition sequence that is complex. Additionally, some delicate reactions must be closely monitored through intermittent sampling in order to bring the reaction to a successful fruition. Where an instrument(s) is capable of performing any of the above, it is still the case that throughput is limited, further, such instruments are extremely expensive.
One prior art attempt at providing an apparatus for the simultaneous, multiple synthesis of organic compounds is found in U.S. Pat. No. 5,324,483 issued Jun. 28, 1994 to Cody et al. In Cody, which is directed toward solid phase synthesis, the lower ends of a plurality of reaction tubes (in the nature of conventional gas dispersion tubes) are each received by a plurality of reaction wells. The reaction tubes are held vertically in place by a holder block, while the reactions wells are contained by a reservoir block. A seal is provided between the holder block and the reservoir block. A manifold covers the reaction tubes, with a seal being provided between the manifold and the holder block. Means are provided for detachably fastening together the reservoir block to the holder block and the holder block to the manifold. The dispersion tubes provide a glass frit type of filtering capability so that a solid support may be retained and rinsed as necessary after performance of a coupling reaction or cleavage of product from the support.
The invention of Cody offers the advantage that multiple reactions may be carried out simultaneouslyxe2x80x94even at reflux at atmospheric pressure conditions and while under an inert atmosphere, if necessary. However, the apparatus appears to be rather complex, bulky, awkward to use, fragile, and difficult to clean. The apparatus also is not very amenable to use with standard liquid handling systems and other batch-processing type equipment. Additionally, the apparatus is not well-suited for true microscale synthesis because the surface area of the components, particularly the gas dispersion tubes, is such that undesirable xe2x80x9chold-upxe2x80x9d of liquid material is prone to occur.
Shown in an article by Meyers et al. entitled xe2x80x9cMultiple simultaneous synthesis of phenolic libraries,xe2x80x9d Molecular Diversity, 1 (1995), pp. 13-20, is an apparatus for multiple, simultaneous (solid phase) synthesis that is considerably better suited to use with batch processing techniques. As described at p. 16 of the article (FIG. 4), Meyers uses a conventional xe2x80x9cdeep-wellxe2x80x9d multi-well plate in the standardized 96-well, 8xc3x9712 rectangular array format. Each well is modified by drilling a small hole in the well bottoms and then installing a filter frit in each well bottom. The deepwell plate is made to be liquid tight by sandwiching it between a clamping arrangement that utilizes a solid base element fitted with four threaded steel posts and a (open) frame element that sits on top of the plate and which is secured by knurl nuts. A planar rubber gasket, which rests on top of the base element, seals the openings at the bottoms of the wells, while the tops of the wells are sealed with 8-well strip caps (a total of twelve such strips would be required for sealing all of the wells). The invention provides that reactions may be carried out within the wells, with the solid support upon which the chemistry is conducted able to then be filtered and washed within the same wells by virtue of the fritted nature of the wells and the removable sealing means at the bottom.
As noted in Meyers, the 96-well format is ubiquitous and used in numerous applications. Meyers"" invention is therefore theoretically able to be used in conjunction with automated high-throughput screens and many other types of equipment (e.g., multi-channel pipettors) that are based on that (standard) 96-well format. However, Meyers is limited in several respects. Perhaps most importantly, the sealing arrangement that is used for the bottom of the wells, i.e., the provision of a clamping frame about the periphery of a multiwell plate having a standard footprint, causes the apparatus to lose that footprint because the frame is naturally dimensionally larger than the multi-well plate. Thus, the multi-well plate with the bottom openings sealed (i.e., with the frame attached) cannot be fitted within the holders of such instruments as automated dispensing equipment that are designed to hold an object that is no larger than the size of the multi-well plate itself.
Another problem is that the outlets at the bottom of the wells are simply holes. There is no provision for a directing nozzle or other structure to be present at the bottom of each well that would enable effluent to properly drain into a collection multi-well plate. Cross-contamination and difficulty with collection into small diameter collecting vessels are highly likely with the Meyer""s invention, especially during forced elution with a vacuum manifold.
Yet another problem is that there appears to be no provision made for heating of the contents of the wells. Since a xe2x80x9cframe clampxe2x80x9d is used on top of the multi-well platexe2x80x94xe2x80x9cframexe2x80x9d indicating an open structure that rests about the perimeter of the top surface of the plate, there is nothing holding the strip-caps in place in the tops of the wells other than a friction fit. Accordingly, any significant expansion of solvent within the wells due to heating would force the caps off. Indeed, the only syntheses described in the article are carried out at 25 degrees C. (i.e., room temperature).
Still another problem lies in the use of caps themselves. Although Meyers describes the use of caps that are in the form of xe2x80x9ceight-well strip capsxe2x80x9d (p. 15), this still necessitates the use of twelve such strips, the caps of each of which must be individually pressed into each well to secure a tight seal. In total, 96 caps must be manipulated and dealt with during performance of a reaction sequence involving all of the wells. Not only is this awkward generally, but cross-contamination may occur if material that is splashed onto the caps inadvertently drips into other wells, or when the caps, after temporary removal, are placed back on their respective wells. Further such caps do not function as a self-sealing gasket to allow the syringed introduction of liquid materials under an inert atmosphere.
Because of the limitations associated with presently available apparatus for multiple, simultaneous synthesis, a great need still exists for such apparatus which can be used on a micro scale, which is suitable for both solid phase synthesis and for organic synthesis generally, which may be used in conjunction with batch processing type equipment, and which is simple and convenient to use.
Accordingly, it is an object of the present invention to provide an apparatus for multiple simultaneous chemical and biological synthesis, chromatography, separation, extraction, analysis and cell culturing on a micro scale which is simple to use, and which is more efficient and inherently capable of a greater range of operations than has heretofore been possible.
It is another object of the invention to provide such an apparatus which is suitable for general organic synthesis, including provision for heating, agitation, addition by syringe, and the like.
It is a further object to provide such an apparatus which has a standard microplate dimension and which is compatible with standard liquid handling and other batch processing equipment.
It is another object to provide such an apparatus which has independently removable upper and lower covers for sealing of the tops and bottom of a multiwell container.
It is a further object to provide such an apparatus with independently removable covers capable of sealing the tops and/or bottoms of all of the wells of a multi-well container simultaneously.
It is yet another object to provide such an apparatus which has an expedient and integral filtration capability.
It is yet a further object to provide such an apparatus that is conveniently used with a vacuum manifold for assisted elution.
It is still another object of the present invention to provide a carrier apparatus that is capable of heating and agitating multiple synthesis apparatuses simultaneously.
Briefly, the preferred embodiment of the present invention is a multi-well synthesis and filtration apparatus for performing multiple, simultaneous chemical reactions on a micro scale. The synthesis apparatus is generally comprised of a deep-well synthesis block having a plurality of nozzle-equipped wells fitted with filter disks and dimensionally arrayed in a standard multi-well format, an upper cover, a lower cover, and a pair of first and second sheet gaskets. The width and length of the covers are dimensionally approximately the same as a standard microplate footprint. The first and second sheet gaskets are interposed between the upper and lower covers, respectively, whereupon the attachment of the covers provides for a tight clamping action to seal inlet portions and outlet spouts of the wells for various reaction and manipulative purposes. Apertures in the upper cover provide that the first gasket may be punctured for introduction of materials and/or maintenance of an inert atmosphere via syringe needle.
There is also provided an alternative embodiment of the synthesis block having wells that are double in volume compared to the first embodiment but which is able to use a multi-well collection plate of the same dimensional array. In this embodiment, there are half the number of wells as compared to the original embodiment, with the wells of a rectangular shape having double the width or diameter of the original wells, but with outlet spouts that depend from the block in an off-center fashion relative to the wells. The outlet spouts are situated so as to drain effluent into every other column of wells of the collection plate. A 180 degree turn of the recycled same block or a second block of the same design provides that the remaining columns of wells of the collection plate may be utilized. An inclined bottom surface portion provides that all liquid properly drains to the outlet spouts.
Once a synthesis reaction has been carried out, a vacuum manifold apparatus is provided to forcibly elute liquid material from the wells as necessary for workup and washing. The manifold apparatus includes a base, a removable lid, a first gasket, a second gasket. The base has an open chamber sized to receive a collection plate and an outlet for applying a reduced pressure to the chamber. The lid has an open area bounded by a shelf. The first and second gaskets have a generally rectangular band shape such that the first gasket is interposable between the base top surface and the lid, and the second gasket is interposable between the shelf and the lower surface perimeter of the synthesis block. With the block located upon the shelf, thereby covering the open area of the lid, a vacuum can be created within the chamber to draw material from the wells and into the collection plate.
For actual performance of a synthesis reaction, a carrier apparatus and a modified incubator oven assembly are provided to enable agitation and heating of multiple ones of the synthesis blocks simultaneously. The carrier apparatus is generally comprised of a platform, a plurality of containment arm assemblies, and a rotation assembly. The platform is open and compartmentalized, and further has a dual-sided nature. The rotation assembly includes a shaft which extends from each end of the platform, one end of the shaft having an engagement head. An oven drive mechanism, which includes a slotted bearing cup to receive the engagement head, rotates the shaft. The synthesis apparatuses are held in cage-like fashion upon one or both sides of the platform during rotation by the containment arm assemblies. In the preferred embodiment, heating and agitation of as many as four of the synthesis apparatuses are permitted simultaneously.
An advantage of the present invention is that the upper and lower covers may be independently attached to and removed from the synthesis block as needed. Thus, for example, reagents and materials may be conveniently added to the wells by removal of a single (upper) cover. On the other hand, during such removal, leakage of material from the outlet spouts is prevented by the attached lower cover. A securely attached upper cover causes liquid material to be retained (by a partial vacuum effect) when the lower cover is removed for transfer of the synthesis block to a vacuum manifold for filtration, among many other examples of benefits provided by such an independent cover system.
Another advantage of the invention is that the provision of securely clampable upper and lower covers allows for heating of the wells (or vigorous agitation) without loss of solvent.
A further advantage is that the design of the synthesis block and cover arrangement provides for a footprint of the same size as a standard multi-well plate-with respect to either the block alone, or the block when fully covered, thereby permitting use of standard liquid handling and other batch processing equipment.
Yet another advantage is that the sheet gasket arrangement and an upper cover which has apertures allows for introduction of materials via syringe and also for a gas inlet (via needle-equipped tubing) for provision of an inert atmosphere.
Yet a further advantage is that the synthesis block is integrally moldable, inexpensive, and disposable.
Still another advantage is that the use of individual filter frits, as compared to some prior art multi-well filtering arrangements which use a sandwiched filter sheet, prevents wicking and cross-contamination between wells.
These and other objects and advantages of the present invention will become clear to those skilled in the art in view of the description of the best presently known mode of carrying out the invention as described herein and as illustrated in the several figures of the drawings.